Summary: Chronic infection with hepatitis B virus (HBV) is a major cause of chronic liver disease, cirrhosis and hepatocellular carcinoma (HCC) worldwide. Globally there are an estimated 257 million persons infected with HBV. In the United States, there are 1.25 million affected individuals and the epidemiology of the infection is changing due to immigration of persons from endemic regions. The natural history of chronic hepatitis B (CHB) also appears to be changing with an increasing prevalence of HBeAg negative chronic hepatitis B and recognition of a group of patients with moderate levels of HBV DNA and ALT levels with undetermined natural history. Knowledge of the rate of disease progression among individuals with moderate levels of HBV DNA and ALT levels is unknown. Despite the availability of safe and effective oral nucleoside analogues for treatment of CHB, therapy remains problematic due to the need for prolonged therapy and limited effectiveness and tolerability of the alternate treatment, interferon. Clearance of hepatitis B surface antigen is the desired surrogate endpoint of therapy but is rarely achieved with current therapy. Identifying the optimal regimen, defining when to treat, for how long and when to stop therapy to achieve this endpoint are major unresolved issues. In addition, defining the best parameters to monitor patients both on and off therapy are not clear. Hypotheses/problems addressed: 1) Define the host, viral and environmental factors that determine the natural history and outcome of HBV infection. Criteria for initiation of antiviral therapy depends on the HBeAg status and certain cutoffs of HBV DNA and ALT levels. However, recent data suggest that a growing number of patients have elevations in HBV DNA and ALT levels outside these proposed cut-offs. The natural history of this group of patients and whether they might benefit from treatment is unknown. We have retrospectively identified a cohort of patients with this indeterminate phenotype and have shown that patients with HBV DNA levels >20,000 IU/mL and who are older age (>40 years) have liver disease. Patients with an indeterminate phenotype and these characteristics should undergo liver biopsy and may benefit from anti-viral treatment. The Liver Diseases Branch is participating in a large multicenter study, the Hepatitis B Research Network, to define the natural history of HBeAg positive and negative chronic hepatitis B. This study has enrolled 2,000 patients at 13 North American sites. Longitudinal monitoring of this large cohort has allowed us to identify patients with atypical hepatitis B phenotypes. Two such phenotypes include HBeAg positive patients with low viral load (typically these patients have high viral load) and conversely HBeAg negative patients with high viral load (typically these patients have low viral load). We performed whole genome sequencing to identify mutations that may be associated with viral replication and with HBeAg loss. We showed that a high mutation rate in HBV was associated with spontaneous loss of HBeAg among HBeAg positive patients. These data suggest data suggest that a high mutation rate in immune active patients suggests increased immune selection pressure contributing to HBeAg loss. 2) Develop and evaluate novel, safer and more effective therapies for chronic viral hepatitis. Current therapy of CHB remains less than optimal. Relapse is common if treatment is discontinued after one year in the absence of HBsAg loss. Consequently, nucleos(t)ides must often be administered long-term or indefinitely. However, long-term use is associated with increased risk of side effects and higher costs. Therefore, the focus of current studies is to develop strategies to induce HBsAg loss (functional cure) to permit discontinuation of therapy and improve outcome of the infection. We are taking several approaches to this problem. The first approach is to combine peginterferon alfa with tenofovir compared to tenofovir alone. This is being conducted as part of the Hepatitis B Research Network and is a multicenter trial that enrolled 200 subjects and is ongoing. The second approach is to treat patients in the immunetolerant phase of the infection. Recent data suggest that patients in this phase of the infection may have preserved immune response perhaps due to younger age and small pilot studies suggested a high rate of HBsAg loss with a combination of interferon alfa with a nucleoside analogue. We conducted a trial to evaluate the safety and efficacy of the combination of entecavir and peginterferon in adults in the immunetolerant phase of chronic HBV infection. Unfortunately, none of the patients met the primary endpoint of both HBeAg loss and HBV DNA 1,000 IU/mL 48 weeks following cessation of therapy. As such a lead-in strategy of 8 weeks of entecavir followed by combination peginterferon and entecavir therapy for 40 weeks was found to have limited efficacy in adults in the immunetolerant phase of chronic HBV infection and cannot be recommended. A manuscript has been submitted for publication. Another approach is to add peginterferon to ongoing long-term nucleos(t)ide analogue therapy for a period of 6 month with the premise that long-term nucleos(t)ide analogue therapy may partially restore the host immune response and increase efficacy of peginterferon therapy. This trial will explore the mechanism of action of interferon during therapy of chronic hepatitis B. This trial has enrolled 13 subjects and data analysis is ongoing. 3) Elucidate the viral pathogenesis of HBV infection and assess novel markers of HBV disease activity. The underlying pathogenesis behind the clinical phases of hepatitis B is poorly understood. Determining the presence versus absence of inflammation can be challenging. We hypothesized that circulating microRNA (miRNA) profiles would be unique between states of active inflammatory and quiescent hepatitis B and might provide valuable insight into disease pathobiology. We collected serum samples from the hepatitis B research network with distinct clinical phenotypes and extracted total circulating miRNA and performed Nanostring miR-array. We identified distinct miRNAs associated with active hepatitis compared to quiescent disease. These miRNAs hold promise as biomarkers and as clues to disease pathogenesis. Among the identified miRNAs, several associated with clinical features and miR-194 also correlated with inflammation and fibrosis on liver biopsy. These data suggest that miR-194 might be a useful biomarker in patients with chronic hepatitis B and indeterminate disease activity. Nucleoside analogues (NA) for chronic hepatitis B (CHB) are highly effective but usually require long-term use, which can be associated with adverse effects and high cost. There has been interest in stopping treatment but hepatitis flares and even death have occurred. HBV RNA, a novel HBV serum marker, has been suggested to correlate with activity of cccDNA. Therefore, we postulated that patients on long-term NA who were HBV DNA negative/HBV RNA positive would be at greater risk for withdrawal flares than those who were negative for both markers. We retrospectively assessed HBV RNA levels from stored sera among subjects who were prospectively withdrawn from NAs and monitored for a period of 192 weeks. We demonstrated that hepatitis flares after withdrawal of antiviral therapy were more common among patients with detectable HBV RNA compared to those who were HBV RNA negative. Flares accompanied by HBeAg seroreversion were associated with significantly higher HBV RNA levels compared to those without HBeAg seroreversion. These results show that HBV RNA levels correlate with HBeAg and HBsAg status and may be a useful biomarker to identify patients who may safely withdraw from antiviral therapy.